Vibrio cholerae, the causative agent of the human diarrheal disease cholera, can be considered to have a pathogenic cycle consisting of a highly motile phase outside the host when no virulence factors are produced, and a colonizing phase within the host intestine when high levels of virulence factors are expressed. Little is known about the requirements for colonization or the environmental conditions which induce virulence factor expression. The investigators have recently found that the alternative sigma factor, sigma 54, and a sigma 54-dependent transcriptional activator protein, FlrC, are required for the expression of distinct sets of genes during both phases of the pathogenic cycle. Moreover, FlrC apparently plays a crucial role in this cycle by simultaneously activating motility genes and repressing virulence factor expression. The experiments in this proposal are focused on the characterization of sigma 54 and FlrC-mediated transcription in V. cholerae, given their important role in the cholera pathogenic cycle. Such studies will provide insights into the molecular mechanisms of pathogenicity, which may lead to novel methods for treatment and prevention of cholera, as well as other infectious diseases. Studies of FlrC function are designed to characterize its 1) phosphorylation, ii) enzymatic (ATPase) activity, iii) DNA-binding, iv) cooperative interactions and v) transcriptional activity. A series of genetic experiments are designed to identify the interactions between FlrC and the virulence regulatory proteins that lead to 1) repression of virulence factor expression and 2) repression of motility gene transcription. Finally, FlrC-controlled genes will be identified by first identifying FlrC binding sites, which lie near the genes FlrC controls, utilizing a technique which involves cycles of selection for FlrC-bound chromosomal fragments followed by PCR amplification. Sequencing the DNA surrounding the selected FlrC binding sites will identify those motility and colonization genes under FlrC control, and the mechanism whereby flrC represses virulence factor production will be revealed. Identification of sigma 54 and FlrC-dependent colonization genes and promoters may be useful in the development of live attenuated cholera vaccines, and manipulation of virulence factor repression could have practical applications for anti-cholera therapy.